Infertility and other untoward reproductive and developmental outcomes, such as spontaneous abortion, fetal and neonatal death, birth defects, and genetic susceptibilities to cancer and other diseases, are associated with genetic damage occurring in mammalian germ cells. Test methodologies employing fluorescence in situ hybridization (FISH) to detect structural and numerical chromosome damage in sperm and early embryonic cells of rodents have been developed at Lawrence Livermore National Laboratory (LLNL) under an NIEHS-DOE Interagency agreement. We are participating in the evaluation of these test methods through the conduct of positive control experiments, by providing sperm from NTP bioassays, and by performing analyses of all data. The rodent sperm-FISH assays being developed provide parallel models of similar sperm-FISH assays in humans. They will be used for evaluating the aneugenic and clastogenic potential of environmental chemicals tested in NTP bioassays, as well as for addressing questions about dose response, differential stage sensitivity, the relationship of defects seen in sperm to those transmitted to the early embryo, and other important issues related to health-risks which cannot be addressed in human studies. During this past year, we have successfully developed, in collaboration with Applied Genetics Laboratory an NIEHS-SBIR laboratory, an X-chromosome probe for the rat. This allowed development of a 3-chromosome rat epididymal sperm-FISH assay (r-ESA). An experiment using the r-ESA on sperm collected from NOVP treated F-344 rats has been designed and will be conducted this next year. Frozen sperm samples collected from an NTP rat study on oxazapam will also be evaluated next year. Samples of homogenization-resistent sperm from the NTP Reproductive Assessment by Continuous Breeding contract are being frozen and retained for sperm-FISH analysis. Centromere-telomere probes were developed by AGL for mouse chromosome 2.